The goal of this research is to determine the molecular mechanisms of tissue-specific and developmentally regulated expression of phosphofructokinase (PFK) isozymes during muscle development. PFK catalyzes a key regulatory rate limiting step in glycolysis, which is crucial for ATP production during muscle contraction. Using isozyme-specific antibodies and the full-length cDNA clones for muscle and liver PFK isolated in this laboratory, we observed that mouse C2 myoblasts synthesized liver and brain isozymes, but during differentiation to myotubes the rate of synthesis and mRNA level for muscle isozyme increased sharply. This research will examine in detail the transition of PFK isozyme gene expression during in vitro myogenesis by comparing relative rates of synthesis, steady state mRNA levels, transcription rates and mRNA turnover for brain and liver PFK. DNA clones coding for muscle PFK will be included. The overall structure of the transcription unit intervening sequences and the 5' flanking and 3' flanking regions of the muscle PFK gene will be characterized and compared with that for the liver PFK gene. The putative regulatory DNA sequence of the muscle PFK gene sufficient for the tissue-specific and developmental stage- specific expression during myogenesis will be defined using DNA- mediated gene transfer and fusion genes. The future long term goal is to identify and characterize the trans-acting regulatory factors which mediate the differential expression of PFK isozymes and which be present in a tissue-and developmental stage-specific manner.